重组HDV核酶逆转录病毒的生产及对HBV抑制效率的测定

Production of recombinant Retrovirus containing the HDV ribozyme and activity of HDV ribozymes to trans-cleave HBV mRNA

目的:构建含HDV核酶基因的重组逆转录病毒载体,包装出逆转录病毒实现其在HepG2215细胞中稳定表达,并研究其是否抑制HBV的复制。方法:应用PCR方法扩增HDV核酶基因,克隆入逆转录病毒载体pMSCV/U6中,用脂质体法转染逆转录病毒载体包装细胞,嘌呤霉素筛选稳定表达细胞株,用PCR和药物抗性水平传播分析检测野生型辅助病毒。收获的病毒感染HepG2215细胞,用酶联免疫实验测定对HBV的抑制率。结果:重组逆转录病毒载体的酶切鉴定片段约为73 bp,与预期值一致。脂质体转染包装细胞,嘌呤霉素加压筛选出高病毒滴度(4.0×106CFU/ml)的细胞克隆,且未检测到辅助病毒,对HBV的抑制率最高可达61.56%。结论:成功构建重组逆转录病毒载体,在包装细胞系中能生产出高滴度的逆转录病毒,能有效的抑制HepG2215细胞内HBV的复制,为HBV的基因治疗提供了实验数据。

Objective： To establish a retroviral vector containing the HDV ribozyme and a stable vector production cell line for HBV treatment. Methods： The HDV ribozyme gene was cloned by PCR using ptVHRZ as a template and recombinated with pMSCV/U6, which had Puromycin resistance gene. The recombinant of HDV ribozyme was evaluated by enzyme digestion and sequencing. The pMSCV/U6-HDVRZ was transfected to pack a cell line with lipofectamine 2000. The titer of the recombinant virus in the. supematant was assayed on NIH3T3 cells. The helper virus was tested by both the PCR and a marker rescue assay. The high-fiter cell lines were chosen for culture, and then the cleavage activity of the retroviral containing the HDV ribozyme was studied in HepG2215 cells by ELISA. Results： The retroviral vector containing the HDV ribozyme was successfully established. The vector production cell lines were established. The highest titer was 4.0 × 10^6 CFU/ml. HDV ribozyme gene integration was detected by PCR. The retrovims was free of a helper virus. The levels of HBsAg of ribozyme groups were significantly lower than those of the control group, proving the HDV ribozyme had HBV-specifie cleavage activity in HepG2215 cells. Conclusion： A retroviral vector containing the HDV ribozyme and a high-titer vector production cell line are successfully constructed. The HBV-specific HDV ribozyme can catalyze the sequence-specific RNA, and suppress the replication of HBV. The retrovirus carrying the ribozyme gene has the potential of being further explored in gene therapy for HBV infection.